RESUMO
Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcription factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astrocytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and destabilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteomics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regulate the NFAT signalosome and cAMP-SOCE crosstalk.
Assuntos
Canais de Cálcio , Cálcio , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteína ORAI1/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
Chronic obstructive lung disease (COPD) and lung cancer are both caused by smoking and often occur as comorbidity. The programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) axis is an important canonic immunoregulatory pathway, and antibodies that specifically block PD-1 or PD-L1 have demonstrated efficacy as therapeutic agents for non-small cell lung cancer. The role of the PD-1/PD-L1 axis in the pathogenesis of COPD is unknown. Here, we analyzed the function of the PD-1/PD-L1 axis in preclinical COPD models and evaluated the concentrations of PD-1 and PD-L1 in human serum and bronchoalveolar lavage (BAL) fluids as biomarkers for COPD. Anti-PD-1 treatment decreased lung damage and neutrophilic inflammation in mice chronically exposed to cigarette smoke (CS) or nontypeable Haemophilus influenzae (NTHi). Ex vivo stimulated macrophages obtained from anti-PD-1-treated mice released reduced amounts of inflammatory cytokines. PD-L1 concentrations correlated positively with PD-1 concentrations in human serum and BAL fluids. Lung sections obtained from patients with COPD stained positive for PD-L1. Our data indicate that the PD-1/PD-L1 axis is involved in developing inflammation and tissue destruction in COPD. Inflammation-induced activation of the PD-1 pathway may contribute to disease progression.
Assuntos
Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Neutrófilos/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Doença Pulmonar Obstrutiva Crônica/metabolismo , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Pulmão/patologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Neutrófilos/patologia , Receptor de Morte Celular Programada 1/metabolismo , Estudos Prospectivos , Doença Pulmonar Obstrutiva Crônica/patologiaRESUMO
Aging is an unstoppable process and begins shortly after birth. Each cell of the organism is affected by the irreversible process, not only with equal density but also at varying ages and with different speed. Therefore, aging can also be understood as an adaptation to a continually changing cellular environment. One of these very prominent changes in age affects Ca2+ signaling. Especially immune cells highly rely on Ca2+-dependent processes and a strictly regulated Ca2+ homeostasis. The intricate patterns of impaired immune cell function may represent a deficit or compensatory mechanisms. Besides, altered immune function through Ca2+ signaling can profoundly affect the development of age-related disease. This review attempts to summarize changes in Ca2+ signaling due to channels and receptors in T cells and beyond in the context of aging.
Assuntos
Envelhecimento/imunologia , Canais de Cálcio/imunologia , Sinalização do Cálcio/imunologia , Cálcio/imunologia , Homeostase/imunologia , Linfócitos T/imunologia , Animais , HumanosRESUMO
The melastatin-related transient receptor potential member 7 (TRPM7) is a unique fusion protein with both ion channel function and enzymatic α-kinase activity. TRPM7 is essential for cellular systemic magnesium homeostasis and early embryogenesis; it promotes calcium transport during global brain ischemia and emerges as a key player in cancer growth. TRPM7 channels are negatively regulated through G-protein-coupled receptor-stimulation, either by reducing cellular cyclic adenosine monophosphate (cAMP) or depleting phosphatidylinositol bisphosphate (PIP2) levels in the plasma membrane. We here identify that heterologous overexpression of human TRPM7-K1648R mutant will lead to disruption of protease or purinergic receptor-induced calcium release. The disruption occurs at the level of Gq, which requires intact TRPM7 kinase phosphorylation activity for orderly downstream signal transduction to activate phospholipase (PLC)ß and cause calcium release. We propose that this mechanism may support limiting GPCR-mediated calcium signaling in times of insufficient cellular ATP supply.
Assuntos
Cálcio/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Canais de Cátion TRPM/metabolismo , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Mutação de Sentido Incorreto , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Canais de Cátion TRPM/genética , Trombina/farmacologiaRESUMO
KEY POINTS: Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca2+ ]i (between 122 and 334 nm) and [Ca2+ ]o (between 23 and 625 µm) for efficient cancer cell elimination, well below blood plasma Ca2+ levels. As predicted from these results, partial down-regulation of the Ca2+ channel Orai1 in CTLs paradoxically increases perforin-dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca2+ optimum compatible with this low Ca2+ optimum for efficient cancer cell killing, whereas the Ca2+ optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca2+ ]o . We propose that a partial inhibition of Ca2+ signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination. ABSTRACT: Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to protect the human body against cancer. Ca2+ is a key metabolic factor for lymphocyte function and cancer homeostasis. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity against cancer cells and found that CTLs have a bell-shaped Ca2+ dependence with an optimum for cancer cell elimination at rather low [Ca2+ ]o (23-625 µm) and [Ca2+ ]i (122-334 nm). This finding predicts that a partial inhibition of Orai1 should increase (rather than decrease) cytotoxicity of CTLs at [Ca2+ ]o higher than 625 µm. We tested this hypothesis in CTLs and indeed found that partial down-regulation of Orai1 by siRNA increases the efficiency of cancer cell killing. We found two mechanisms that may account for the Ca2+ optimum of cancer cell killing: (1) migration velocity and persistence have a moderate optimum between 500 and 1000 µm [Ca2+ ]o in CTLs, and (2) lytic granule release at the immune synapse between CTLs and cancer cells is increased at 146 µm compared to 3 or 800 µm, compatible with the Ca2+ optimum for cancer cell killing. It has been demonstrated in many cancer cell types that Orai1-dependent Ca2+ signals enhance proliferation. We propose that a decrease of [Ca2+ ]o or partial inhibition of Orai1 activity by selective blockers in the tumour microenvironment could efficiently reduce cancer growth by simultaneously increasing CTL and NK cell cytotoxicity and decreasing cancer cell proliferation.
Assuntos
Apoptose , Cálcio/metabolismo , Proliferação de Células , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T Citotóxicos/imunologia , Movimento Celular , Grânulos Citoplasmáticos/metabolismo , Humanos , Neoplasias/metabolismo , Perforina/metabolismo , Células Tumorais CultivadasRESUMO
Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg(2+)), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg(2+) ([Mg(2+)]i) and this is facilitated through the ATP-binding site of the channel's kinase domain. The synergistic block of TRPM7 by chloride and Mg(2+) is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg(2+)]i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Brometos/farmacologia , Cloretos/farmacologia , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica/fisiologia , Iodetos/farmacologia , Canais de Cátion TRPM/metabolismo , Brometos/metabolismo , Proliferação de Células/efeitos dos fármacos , Cloretos/metabolismo , DNA Complementar/biossíntese , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Iodetos/metabolismo , Células MCF-7 , Técnicas de Patch-Clamp , Proteínas Serina-Treonina Quinases , Estrutura Terciária de Proteína/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Simportadores/metabolismo , Canais de Cátion TRPM/fisiologiaRESUMO
Abstract Agonist-induced Ca(2+) oscillations in many cell types are triggered by Ca(2+) release from intracellular stores and driven by store-operated Ca(2+) entry. Stromal cell-interaction molecule (STIM) 1 and STIM2 serve as endoplasmic reticulum Ca(2+) sensors that, upon store depletion, activate Ca(2+) release-activated Ca(2+) channels (Orai1-3, CRACM1-3) in the plasma membrane. However, their relative roles in agonist-mediated Ca(2+) oscillations remain ambiguous. Here we report that while both STIM1 and STIM2 contribute to store-refilling during Ca(2+) oscillations in mast cells (RBL), T cells (Jurkat) and human embryonic kidney (HEK293) cells, they do so dependent on the level of store depletion. Molecular silencing of STIM2 by siRNA or inhibition by G418 suppresses store-operated Ca(2+) entry and agonist-mediated Ca(2+) oscillations at low levels of store depletion, without interfering with STIM1-mediated signals induced by full store depletion. Thus, STIM2 is preferentially activated by low-level physiological agonist concentrations that cause mild reductions in endoplasmic reticulum Ca(2+) levels. We conclude that with increasing agonist concentrations, store-operated Ca(2+) entry is mediated initially by endogenous STIM2 and incrementally by STIM1, enabling differential modulation of Ca(2+) entry over a range of agonist concentrations and levels of store depletion.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Drosophila , Retículo Endoplasmático/metabolismo , Inativação Gênica , Gentamicinas/farmacologia , Células HEK293 , Humanos , Mastócitos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , RNA Interferente Pequeno , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Linfócitos T/metabolismoRESUMO
TRPM3 channels form ionotropic steroid receptors in the plasma membrane of pancreatic ß and dorsal root ganglion cells and link steroid hormone signaling to insulin release and pain perception, respectively. We identified and compared the function of a number of TRPM3 splice variants present in mouse, rat and human tissues. We found that variants lacking a region of 18 amino acid residues display neither Ca(2+) entry nor ionic currents when expressed alone. Hence, splicing removes a region that is indispensable for channel function, which is called the ICF region. TRPM3 variants devoid of this region (TRPM3ΔICF), are ubiquitously present in different tissues and cell types where their transcripts constitute up to 15% of the TRPM3 isoforms. The ICF region is conserved throughout the TRPM family, and its presence in TRPM8 proteins is also necessary for function. Within the ICF region, 10 amino acid residues form a domain essential for the formation of operative TRPM3 channels. TRPM3ΔICF variants showed reduced interaction with other TRPM3 isoforms, and their occurrence at the cell membrane was diminished. Correspondingly, coexpression of ΔICF proteins with functional TRPM3 subunits not only reduced the number of channels but also impaired TRPM3-mediated Ca(2+) entry. We conclude that TRPM3ΔICF variants are regulatory channel subunits fine-tuning TRPM3 channel activity.
Assuntos
Processamento Alternativo , Canais de Cátion TRPM/genética , Sequência de Aminoácidos , Animais , Sinalização do Cálcio , Sequência Conservada , Éxons , Células HEK293 , Humanos , Imunoprecipitação , Potenciais da Membrana , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sítios de Splice de RNA , Ratos , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismoRESUMO
Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Cálcio/deficiência , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Quelantes/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Inositol 1,4,5-Trifosfato/farmacologia , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Células Jurkat , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteína ORAI1 , Molécula 1 de Interação Estromal , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Numerous alterations in CD8+ T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell-intrinsic dysfunctions and microenvironmental changes is challenging. TCR transgenic OT-I mice are utilized to investigate CD8+ T-cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT-I model minimizes the current limitations and provides a valuable tool to assess antigen-specific T-cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8+ T cells from OT-I+/+ and OT-I+/- mice to prove the applicability of the heterozygous system. Our data reveal that OVA-activated CD8+ T cells from adult OT-I+/- mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age-related alterations in CD8+ T cells, including naive T-cell deterioration and decreased proliferative capacity, also occur in elderly OT-I+/- mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT-I+/- model to investigate cell-intrinsic alterations affecting the cytotoxic behavior of aged CD8+ T cells after antigen-specific in vitro activation. Time-resolved analysis of antigen-directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8+ T cells increases with age. Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8+ T cells shifts the mode of target cell death from granzyme-mediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation.
Assuntos
Antígenos , Linfócitos T CD8-Positivos , Camundongos , Animais , Camundongos Transgênicos , Regulação para Cima , Necrose , Camundongos Endogâmicos C57BL , OvalbuminaRESUMO
TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic ß cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents.
Assuntos
Membrana Celular/metabolismo , Canais de Cátion TRPM/metabolismo , Zinco/farmacologia , Linhagem Celular , Eletrofisiologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunoprecipitação , Mutação , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPM/genéticaRESUMO
Plasma membrane protein channels provide a passageway for ions to access the intracellular milieu. Rapid entry of calcium ions into cells is controlled mostly by ion channels, while Ca2+-ATPases and Ca2+ exchangers ensure that cytosolic Ca2+ levels ([Ca2+]cyt) are maintained at low (~100 nM) concentrations. Some channels, such as the Ca2+-release-activated Ca2+ (CRAC) channels and voltage-dependent Ca2+ channels (CACNAs), are highly Ca2+-selective, while others, including the Transient Receptor Potential Melastatin (TRPM) family, have broader selectivity and are mostly permeable to monovalent and divalent cations. Activation of CRAC channels involves the coupling between ORAI1-3 channels with the endoplasmic reticulum (ER) located Ca2+ store sensor, Stromal Interaction Molecules 1-2 (STIM1/2), a pathway also termed store-operated Ca2+ entry (SOCE). The TRPM family is formed by 8 members (TRPM1-8) permeable to Mg2+, Ca2+, Zn2+ and Na+ cations, and is activated by multiple stimuli. Recent studies indicated that SOCE and TRPM structure-function are interlinked in some instances, although the molecular details of this interaction are only emerging. Here we review the role of TRPM and SOCE in Ca2+ handling and highlight the available evidence for this interaction.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio , Canais de Cátion TRPM , Canais de Potencial de Receptor Transitório , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Proteína ORAI1/metabolismoRESUMO
A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8+ T cells responses in the elderly has come back into focus since the COVID-19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8+ T cells in aging. We focused on the different subpopulations and time-resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8+ T cells from elderly mice are much faster than those of CD8+ T cells from adult mice. This is true not only in the total CD8+ T cell population but also for their effector (TEM ) and central memory (TCM ) T cell subpopulations. TIRF experiments reveal that CD8+ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8+ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell-intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked.
Assuntos
COVID-19 , Neoplasias , Animais , Linfócitos T CD8-Positivos , Citotoxicidade Imunológica , Granzimas , Humanos , Glicoproteínas de Membrana , Camundongos , Pandemias , Perforina , Proteínas Citotóxicas Formadoras de PorosRESUMO
STIM1 in the endoplasmic reticulum and CRACM1 in the plasma membrane are essential molecular components for controlling the store-operated CRAC current. CRACM1 proteins multimerize and bind STIM1, and the combined overexpression of STIM1 and CRACM1 reconstitutes amplified CRAC currents. Mutations in CRACM1 determine the selectivity of CRAC currents, demonstrating that CRACM1 forms the CRAC channel's ion-selective pore, but the CRACM1 homologs CRACM2 and CRACM3 are less well characterized. Here, we show that both CRACM2 and CRACM3, when overexpressed in HEK293 cells stably expressing STIM1, potentiate I(CRAC) to current amplitudes 15-20 times larger than native I(CRAC). A nonconducting mutation of CRACM1 (E106Q) acts as a dominant negative for all three CRACM homologs, suggesting that they can form heteromultimeric channel complexes. All three CRACM homologs exhibit distinct properties in terms of selectivity for Ca(2+) and Na(+), differential pharmacological effects in response to 2-APB, and strikingly different feedback regulation by intracellular Ca(2+). Each of the CRAC channel proteins' specific functional features and the potential heteromerization provide for flexibility in shaping Ca(2+) signals, and their characteristic biophysical and pharmacological properties will aid in identifying CRAC-channel species in native cells that express them.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Compostos de Boro , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular , Humanos , Potenciais da Membrana/fisiologia , Mutação/genética , Proteína ORAI1 , Molécula 1 de Interação EstromalRESUMO
Ca2+ is a crucial second messenger for proper T cell function. Considering the relevance of Ca2+ signals for T cell functionality it is surprising that no mechanistic insights into T cell Ca2+ signals from elderly individuals are reported. The main Ca2+ entry mechanism in T cells are STIM-activated Orai channels. Their role during lymphocyte aging is completely unknown. Here, we report not only reduced Ca2+ signals in untouched and stimulated, but also in central and effector memory CD8+ T cells from elderly (18-24 months) compared to adult (3-6 months) mice. Two mechanisms contribute to the overall reduction in Ca2+ signals of CD8+ T cells of elderly mice: 1) Reduced Ca2+ currents through Orai channels due to decreased expressions of STIMs and Orais. 2) A faster extrusion of Ca2+ owing to an increased expression of PMCA4. The reduced Ca2+ signals correlated with a resistance of the cytotoxic efficiency of CD8+ T cells to varying free [Ca2+]ext with age. In summary, reduced STIM/Orai expression and increased Ca2+ clearing rates following enhanced PMCA4 expression contribute to reduced Ca2+ signals in CD8+ T cells of elderly mice. These changes are apparently relevant to immune function as they reduce the Ca2+ dependency of CTL cytotoxicity.
Assuntos
Envelhecimento/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Sinalização do Cálcio/fisiologia , Proteína ORAI1/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Cálcio/metabolismo , Camundongos , Proteína ORAI1/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Molécula 1 de Interação Estromal/genéticaRESUMO
Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca(2+) binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.
Assuntos
Cálcio/metabolismo , Transporte de Íons/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Western Blotting , Canais de Cálcio , Linhagem Celular , Clonagem Molecular , Ácido Glutâmico/genética , Glutamina/genética , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Proteína ORAI1 , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Alinhamento de Sequência , Molécula 1 de Interação EstromalRESUMO
STIM1 and CRACM1 (or Orai1) are essential molecular components mediating store-operated Ca2+ entry (SOCE) and Ca2+ release-activated Ca2+ (CRAC) currents. Although STIM1 acts as a luminal Ca2+ sensor in the endoplasmic reticulum (ER), the function of STIM2 remains unclear. Here we reveal that STIM2 has two distinct modes of activating CRAC channels: a store-operated mode that is activated through depletion of ER Ca2+ stores by inositol 1,4,5-trisphosphate (InsP3) and store-independent activation that is mediated by cell dialysis during whole-cell perfusion. Both modes are regulated by calmodulin (CaM). The store-operated mode is transient in intact cells, possibly reflecting recruitment of CaM, whereas loss of CaM in perfused cells accounts for the persistence of the store-independent mode. The inhibition by CaM can be reversed by 2-aminoethoxydiphenyl borate (2-APB), resulting in rapid, store-independent activation of CRAC channels. The aminoglycoside antibiotic G418 is a highly specific and potent inhibitor of STIM2-dependent CRAC channel activation. The results reveal a novel bimodal control of CRAC channels by STIM2, the store dependence and CaM regulation, which indicates that the STIM2/CRACM1 complex may be under the control of both luminal and cytoplasmic Ca2+ levels.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/fisiologia , Calmodulina/fisiologia , Moléculas de Adesão Celular/fisiologia , Proteínas de Membrana/fisiologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Calmodulina/genética , Moléculas de Adesão Celular/genética , Linhagem Celular , Citoplasma/fisiologia , Humanos , Proteínas de Membrana/genética , Proteína ORAI1 , Molécula 2 de Interação Estromal , Regulação para CimaRESUMO
The tumor microenvironment (TME) is shaped by cancer and noncancerous cells, the extracellular matrix, soluble factors, and blood vessels. Interactions between the cells, matrix, soluble factors, and blood vessels generate this complex heterogeneous microenvironment. The TME may be metabolically beneficial or unbeneficial for tumor growth, it may favor or not favor a productive immune response against tumor cells, or it may even favor conditions suited to hijacking the immune system for benefitting tumor growth. Soluble factors relevant for TME include oxygen, reactive oxygen species (ROS), ATP, Ca2+, Hâº, growth factors, or cytokines. Ca2+ plays a prominent role in the TME because its concentration is directly linked to cancer cell proliferation, apoptosis, or migration but also to immune cell function. Stromal-interaction molecules (STIM)-activated Orai channels are major Ca2+ entry channels in cancer cells and immune cells, they are upregulated in many tumors, and they are strongly regulated by ROS. Thus, STIM and Orai are interesting candidates to regulate cancer cell fate in the TME. In this review, we summarize the current knowledge about the function of ROS and STIM/Orai in cancer cells; discuss their interdependencies; and propose new hypotheses how TME, ROS, and Orai channels influence each other.
RESUMO
Chronic obstructive pulmonary disease (COPD) is associated with neutrophilic lung inflammation and CD8 T cell exhaustion and is an important risk factor for the development of non-small cell lung cancer (NSCLC). The clinical response to programmed cell death-1 (PD-1) blockade in NSCLC patients is variable and likely affected by a coexisting COPD. The pro-inflammatory cytokine interleukin-17C (IL-17C) promotes lung inflammation and is present in human lung tumors. Here, we used a Kras-driven lung cancer model to examine the function of IL-17C in inflammation-promoted tumor growth. Genetic ablation of Il-17c resulted in a decreased recruitment of inflammatory cells into the tumor microenvironment, a decreased expression of tumor-promoting cytokines (e.g. interleukin-6 (IL-6)), and a reduced tumor proliferation in the presence of Haemophilus influenzae- (NTHi) induced COPD-like lung inflammation. Chronic COPD-like inflammation was associated with the expression of PD-1 in CD8 lymphocytes and the membrane expression of the programmed death ligand (PD-L1) independent of IL-17C. Tumor growth was decreased in Il-17c deficient mice but not in wildtype mice after anti-PD-1 treatment. Our results suggest that strategies targeting innate immune mechanisms, such as blocking of IL-17C, may improve the response to anti-PD-1 treatment in lung cancer patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Imunidade Inata , Interleucina-17/fisiologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Animais , Antígeno B7-H1/biossíntese , Antígeno B7-H1/genética , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/etiologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Linhagem Celular Tumoral , Citocinas/fisiologia , Feminino , Genes ras , Humanos , Interleucina-17/deficiência , Interleucina-17/genética , Interleucina-17/farmacologia , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neutrófilos/fisiologia , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/genética , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/imunologia , Proteínas Recombinantes/farmacologia , Microambiente TumoralRESUMO
2-Aminoethoxydiphenyl borate (2-APB) has emerged as a useful pharmacological tool in the study of store-operated Ca(2+) entry (SOCE). It has been shown to potentiate store-operated Ca(2+) release-activated Ca(2+) (CRAC) currents at low micromolar concentrations and to inhibit them at higher concentrations. Initial experiments with the three CRAC channel subtypes CRACM1, CRACM2 and CRACM3 have indicated that they might be differentially affected by 2-APB. We now present a thorough pharmacological profile of 2-APB and report that it can activate CRACM3 channels in a store-independent manner without the requirement of STIM1, whereas CRACM2 by itself is completely unresponsive to 2-APB and CRACM1 is only very weakly activated. However, when coexpressed with STIM1 and activated via store depletion, CRACM1 and CRACM2 are facilitated at low 2-APB concentrations and inhibited at higher concentrations, while CRACM3 only exhibits potentiated currents. Consistently, the 2-APB-induced CRAC currents exhibit altered selectivities that are characterized by a leftward shift in reversal potential and the emergence of large outward currents that are carried by normally impermeant monovalent cations such as Cs(+) or K(+). These results suggest that 2-APB has agonistic and antagonistic modes of action on CRAC channels, acting at the channel level as a store-independent and direct gating agonist for CRACM3 and a potentiating agonist for CRACM1 and CRACM2 following store-operated and STIM1-dependent activation. The inhibition of CRACM1 channels by high concentrations of 2-APB appears to involve a direct block at the channel level and an additional uncoupling of STIM1 and CRACM1, since the compound reversed the store-dependent multimerization of STIM1. Finally, we demonstrate that single-point mutations of critical amino acids in the selectivity filter of the CRACM1 pore (E106D and E190A) enable 2-APB to gate CRACM1 in a STIM1-independent manner, suggesting that 2-APB facilitates CRAC channels by altering the pore architecture.